In recent years there has been much development of liposomes comprising functional molecules introduced into the surface of the liposome membrane as vectors for delivering drugs, nucleic acids, proteins, sugars or other substances to target sites.
For example, liposomes have been developed comprising hydrophilic polymers (for example, polyalkylene glycols such as polyethylene glycol) introduced into the outer surface of the liposome membrane (Japanese Patent Applications Laid-open Nos. H1-249717, H2-149512, H4-346918, 2004-10481). With these liposomes, it is possible to improve the directionality of the liposome for tumor cells by improving the retention of the liposome in blood.
Moreover, liposomes have been developed having introduced into the outer surface of the liposome membrane a substance (such as transferrin, insulin, folic acid, hyaluronic acid, an antibody or fragment thereof or a sugar chain) capable of binding to a receptor or antigen present on the surface of the cell membrane (Japanese Patent Applications Laid-open Nos. H4-346918, 2004-10481). With these liposomes it is possible to improve the endocytosis efficiency of the liposome.
Liposomes have also been developed using cholesterol bound to GALA in which GALA is introduced into the outer surface of the liposome membrane (T. Kakudo et al., Biochemistry, 2004, Vol. 43, pp. 5618 to 5623). A liposome becomes enveloped by endosome in the process of endocytosis, and inside the endosome the liposome is broken down when the endosome fuses with the lysosome, but with this kind of liposome the liposome-entrapped substance can escape from the endosome and be released into the cytoplasm.
GALA is a synthetic peptide comprising the amino acid sequence represented by SEQ ID NO:1, which was synthesized by the group of Szoka et al (N. K. Subbarao et al, Biochemistry, 1987, Vol. 26, pp. 2964 to 2972) and has been much studied since then.
GALA is pH sensitive, assuming a random coil structure at a pH of 7.4, but when the pH rises to about 5.0 the charge of the glutamic acid residue is neutralized, extinguishing the electrical repulsion and producing an α helix structure (N. K. Subbarao et al., Biochemistry, 1987, Vol. 26, pp. 2964 to 2972). The proportion of a helix structures is about 20% at pH 7.4 but rises to about 70% when the pH rises to 5.0 (E. Goormaghtigh et al., European Journal of Biochemistry, 1991, Vol. 195, pp. 421 to 429.
When GALA is incubated under acidic conditions with liposomes comprising egg-yolk phosphatidylcholine, the liposome-entrapped substance leaks out (an effect which is strongest at pH 5.0), and the liposomes fuse with one another (R. A. Parente et al., Journal of Biological Chemistry, 1988, Vol. 263, pp. 4724 to 4730). Regarding the mechanism by which GALA causes the release of the liposome-entrapped substance, the suggestion is that when GALA penetrates the liposome membrane, the GALA penetrating the liposome membrane clumps in groups of 8 to 12 in the membrane, forming pores 5 to 10 Å in diameter (R. A. Parente et al., Biochemistry, 1990, Vol. 29, pp. 8720 to 8728).